Cystic Fibrosis (CF) is the most common life-shortening autosomal recessive condition in the Caucasian population, with an estimated 25,000 people affected and seven million carriers in the U.S. alone.
Over the last 29 years, the median age of survival of people having CF has increased dramatically from 14 years in 1969 to 31 years of age in 1997. CF is therefore now both a pediatric and adult disease. Growth and nutritional status in people having CF are related to the severity of lung disease, the severity of pancreatic insufficiency, and to nutrient intake and absorption.
Considering national data (CFF Patient Registry), nutrition-related growth failure is still at an unacceptably high rate. In 1997, 19% of patients having CF were below the 5th percentile for height, and 24% were below the 5th percentile for weight. If the 10th percentile for weight is considered the more appropriate screen for patients with a nutritionally high-risk condition such as CF, then 33% of the patients with CF were below the desirable level in 1997. Except in the end stage of the lung disease, most of this growth failure and poor nutritional status (reduced weight for height) is associated with pancreatic insufficiency.
Improved treatment of pancreatic insufficiency in CF patients, as well as in other patients suffering from non-CF related malabsorption, requires an efficient clinical care test for malabsorption of nutrients through the intestinal wall. New pancreatic enzyme products or modifications of current products require evaluation in a research quality test for malabsorption. Given the occurrence of the previously unrecognized diagnosis of fibrosing colonopathy, accurate documentation of the severity of malabsorption is even more important to the CF care community and to patients having CF and pancreatic insufficiency (Smyth R. L., et al., 1995; Lancet, 346(8985):1247-51; Lloyd-Still, J. D., et al., 1998; J. Pediatr Gastroenterol Nutr., 26(5):489-95).
The important link between pancreatic insufficiency and malnutrition in children with CF was established over 40 years ago (Pencharz, P. B. et al., 1993; Annu. Rev. Nutr. 13:111-36). These investigators demonstrated that pancreatic enzymes improved fat absorption and led to positive nitrogen balance in the patient. Steatorrhea and malabsorption are the presenting symptoms in more than 20% of infants and children with CF. More than 85% of individuals with CF have pancreatic insufficiency and require exogenously added pancreatic enzymes in their diet (FitzSimmons, S. C., 1993; J. Pediatr., 122:1-9). Assessing the degree of fat malabsorption in the intestine is helpful for guiding both enzyme therapy and nutritional intervention in children and adults having CF and pancreatic insufficiency.
The are a variety of other pathological conditions that are associated with aberrant fat absorption. These include, without limitation, hereditary pancreatitis, α1 anti-trypsin deficiency, Shwachman Syndrome, Johanson-Blizzard Syndrome, sideroblastic anemia, pancreatic insufficiency, lipase deficiency, co-lipase deficiency in children, both partial and complete pancreatic surgical resection, pancreatic cancer, chronic and autoimmune pancreatitis, hyperlipidemia, and hyperparathyroidism. Improved methods for assessing aberrant pancreatic-based fat malabsorption in each of these disorders should facilitate diagnosis and the development of disease management regimens.
The 72-hour stool and diet collection method is considered the standard test for measuring the degree of fat malabsorption in a patient (Van De Kamer, J. H. et al., 1949; J. Biol Chem. 177:345-55; Jeejeebhoy, K. N. et al., 1970; Clin. Biochem. 3:157-63). A coefficient of fat absorption (CFA) obtained from the 72-hour stool collection and dietary intake method is widely accepted as a clinical, diagnostic, but non-specific, test for assessing fat malabsorption. The accuracy of this test is dependent on at least three days of entire stool collections together with consumption and documentation of a defined moderate to high fat diet. Despite its chemical accuracy, this test is prone to errors due to inadequate documentation of dietary intake, incomplete stool collections and day-to-day variation in fecal fat excretion (Holmes, G. K. T., 1988; BMJ, 296:1552). The technical and aesthetic difficulties associated with stool collection, storage and analysis make this test very unappealing to patients, families and lab workers. Few local laboratories conduct quantitative fecal fat analyses, thereby requiring additional time and expense for stools to be shipped to regional labs. The lag period between sample collection and reported results further fuels the disinterest in this test. Therefore, despite the clinically important data provided by the 72-hour stool and dietary collection method, this test is under-utilized or avoided in most clinical care and research settings. Even with the availability of capable labs, this potentially important clinical test is poorly accepted and under-utilized by both patients and care providers (Holmes, G. K. T., 1988; BMJ, 296:1552) thus contributing to the malnutrition and growth failure associated with CF.
Several alternative tests have been developed for assessing fat malabsorption, but all have significant limitations and have failed to gain wide acceptance as accurate or practical alternatives to the 72-hour stool and diet collection method. Several of these tests are discussed below.
The Spot Sudan III stain test is a qualitative test which involves the microscopic examination of a random fecal specimen stained with the Sudan dye (Drummey, G. D., et al., 1961; N. Engl. J. Med., 264:85-7). Even when objective criteria are used, the results are poorly reproducible in the ranges of mild to moderate steatorrhea common in patients with CF. The accuracy of this test is increased when homogenized pooled stool samples are used. However, from the patient's and family's perspective, this renders the test no different from the 72-hour stool collection method.
The Acid Steatocrit test utilizes a small (about 5 grams) stool specimen which is spun to separate the acidified fat from the solid stool (Tran, M., et al., 1994; J. Pediatr. Gastroenterol Nutr., 19:299-303; Phuapradit P., et al., 1981; Arch. Dis. Child., 56:725-7). Correlation with the 72-hour stool and diet collection method only occurs when homogenized-pooled stool is used. Therefore, requirement for a pooled stool collection offers no added benefit to the patient and family, and is not well correlated with the 72-hour collection method.
Breath tests, such as 14C and 13C Triolein breath tests (Newcomer, A. D., et al., 1979; Gastroenterology, 76:6-13; Watkins, J. B., et al., 1982; Gastroenterology, 82:911-7), are non-invasive tests utilizing small amounts of dietary fat labeled with stable or radioactive isotopes. Fat malabsorption is predicted by the labeled CO2, and is an indicator of the absorbed dietary fat. The data may be used to qualitatively monitor response to pancreatic enzyme therapy in cases of known pancreatic insufficiency (Amarri, S., et al., 1997; Arch. Dis. Child., 76:349-51; Goff, J. S., 1982; Gastroenterology, 83:44-46). When compared with 72-hour stool and diet collection method, breath tests had up to 80% sensitivity but only 45% specificity (Pedersen, N. T., et al., 1991; Scand. J. Clin. Lab. Invest. 51:699-703). In using the breath test, there is also the assumption that the subject's pulmonary status is normal or near normal, which is not always the case in patients having CF. Also, 14C is a radioactive compound, and therefore not suitable for use in children. 13C, the stable isotope, is safe for use in children, however, costs of the substrate and equipment required for analysis (mass spectrometry) are very high.
Retinyl palmitate, a long chain fat which is hydrolyzed by lipase prior to intestinal absorption via the chylomicron route, has successfully been used as marker for monitoring postprandial chylomicron response to dietary fat. However, the specificity of retinyl palmitate for assessing fat malabsorption due to pancreatic insufficiency in humans is limited because there are intrinsic gut brush border lipase enzymes which specifically hydrolyze retinyl esters (Rigtrup, K. M., et al., 1994; Am. J. Clin. Nutr., 60:111-116). In addition, specific defects involving the intestinal handling of retinol may occur in CF and other pancreatic-fat based malabsorption disorders, further limiting the use of this assay to monitor dietary fat malabsorption caused by pancreatic insufficiency (Ahmed, F., et al., 1990; Arch. Dis. Child., 65(6):589-593).
Apolipoprotein A-IV (A-4) is a 46-kD lipoprotein exclusively synthesized with chylomicrons in the enterocyte in response to dietary fat in humans (Green, P. H. R., et al., 1982; Gastroenterology, 83:1223-30). The rate of synthesis and secretion of this compound correlates with the consumption of dietary long chain triglycerides in a dose dependent pattern (Rodriguez, M-D., et al., 1997; Am. J. Physiol., 272:R1170-177). Since A-4 is rapidly catabolized, serum levels of this compound are dependent on rate of synthesis of A-4, and therefore A-4 may be used as a marker of absorbed dietary fat (Rader, D. J., et al., 1993; J. Clin. Invest., 92:1009-17). However, the immunoassays used for the detection of A-4 are not well standardized, thereby limiting broad, clinical utility.
The Pentadecanoic Acid (IPPA) test is a non-invasive qualitative test in adults used to evaluate pancreatic lipase activity. The assay involves oral administration of a radiolabeled triglyceride containing iodophenylpentadecanoic acid, 123I-IPPA, as a long chain fatty acid linked to position-3 of the glycerol backbone. Hydrolysis of the triglyceride by pancreatic lipase is required before IPPA is absorbed. After absorption, IPPA is metabolized by β-oxidation and the final metabolic product, 123I-p-iodobenzoic acid, is excreted in urine. Scintigraphic analysis of urine for 123I-p-iodobenzoic acid provides qualitative data about the activity of pancreatic lipase. However, the involvement of radioactivity limits broad applicability of this test to children.
Other tests which analyze stool for fat and energy content have been developed, including fecal bomb calorimetry and near infrared fecal spectroscopy (Benini, L., et al., 1989; Gut., 30:1344-7; Emmanuel, B., 1974; Biochem. Biophys. Acta., 337:404-13). In all cases, correlation with the 72-hour stool and diet collection method only occurs when pooled homogenized fecal samples are used. Therefore, these tests provide no added benefit to the patient and family than those described above.
All of the prior art assays have failed to become widely accepted in CF research or clinical care of patients with pancreatic fat-based malabsorption disorders. Thus, there remains a need in the art for improvements in methods for the diagnosis and nutritional care of patients with CF and other pancreatic insufficiency-related pathologies.